Compound B as angiogenic agent in combination with human growth factors

ABSTRACT

The present invention refers to the use of Compound B as angiogenic agent in combination with human growth factors.

FIELD OF THE INVENTION

[0001] The present invention refers to the use of Component B asangiogenic agent in combination with human growth factors.

STATE OF THE ART

[0002] Component B (hereinafter indicated as CB) is a 81 amino acidprotein originally isolated from human urine. The human gene expressingthe protein has been cloned and expressed in CHO cells as recombinanthuman Component B, the protein has a molecular weight of about 8.9 kDand was thoroughly described in WO 94/14259 to which reference is madealso for the methods of preparation and its amino acid sequence.

[0003] In WO97/39765 the use of CB as cicatrizant was described.

[0004] It is also known that growth factors, as for example basicfibroblast growth factor (bFGF) or vascular endothelial growth factor(VEGF) have angiogenic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005]FIG. 1: Shows the implant of a pellet loaded with both activecompounds (1A) or of two pellets each loaded with one active compound (1B) in the rabbit cornea.

[0006]FIG. 2: Shows the effect of bFGF and CB on angiogenesis in therabbit cornea.

[0007]FIG. 3: Shows the effect of heat-inactivation on the angiogenicactivity of CB.

[0008]FIG. 4: Shows the synergistic effect of CB on bFGF-inducedangiogenesis.

[0009]FIG. 5: Shows the synergistic effect of CB on the angiogenicactivity of bFGF.

[0010]FIG. 6: Represents a drawing of a typical histological section ofrabbit cornea illustrating the main structures observable in followingFIGS. 7-11.

[0011]FIG. 7: section of rabbit cornea (x200) with a corneal pocketcontaining 100 ng of bFGF sampled at 6 days post-surgery wherein thearrows show neoformed vessels. (Ep=epithelium).

[0012]FIG. 8: section of rabbit cornea (x100) with a corneal pocketcontaining 500 ng of CB sampled at 2 days post-surgery wherein thearrows show neoformed vessels.

[0013]FIG. 9: section of rabbit cornea (x 200) with a corneal pocketcontaining 500 ng of CB sampled at 6 days post-surgery wherein thearrows show neoformed vessels.

[0014]FIG. 10: Section of rabbit cornea (x100) with a corneal pocketcontaining 4 μg of CB sampled at 15 days post-surgery wherein the arrowshow neoformed vessels.

[0015]FIG. 11: Section of rabbit cornea (x100) with a corneal pocketcontaining 500 ng of bFGF sampled at 15 days post-surgery wherein thearrow show neoformed vessels.

[0016]FIG. 12: mean and 95.0% Tukey HSD Intervals

DETAILED DESCRIPTION OF THE INVENTION

[0017] It was now surprisingly found that the simultaneous presence ofCB and a growth factor increases the angiogenic response elicited byeither individual agent, in other words CB is capable of synergizingwith a growth factor in promoting neovascular growth in the tissuesprobably facilitating some early events required to mobilise endothelialcells from capillaries.

[0018] Therefore, the main object of the present invention is the use ofCB in combination with a growth factor for the manufacture ofpharmaceutical compositions useful for the treatment of wounds, ulcersand other traumatic lesions to any of the tissues in the body.

[0019] Another object of the invention are pharmaceutical compositionsprepared as described above.

[0020] A further object of this invention is a method of treatment ofwounds, ulcers and other traumatic lesions to any of the tissues in thebody, comprising administering an effective amount of CB and of a growthfactor, possibly together with a pharmaceutically acceptable excipient.

[0021] The administration of the active ingredient may be oral,intravenous, intramuscular, subcutaneous or topical. Other routes ofadministration, which may establish the desired blood levels ofrespective active agents are comprised by the present invention.

[0022] The administration of the two active compounds can be performedby a single pharmaceutical preparation containing both of them or,preferably, by two pharmaceutical preparation each containing separatelyone of the two ingredients. Preferred growth factor to be used incombination with CB according to the invention are the basic fibroblastgrowth factor (bFGF) or the vascular endothelial growth factor (VEGF).

[0023] Angiogenesis was studied in the cornea of albino rabbits sincethis is an avascular and transparent tissue where inflammatory reactionsand growing capillaries can be easily monitored and changes quantifiedby stereomicroscopic examination (Ziche et al., 1982). This methodallows the monitoring over an extended period of time of vessel growthby direct and non traumatic observation of the process. Moreover in thesame animal the quantification of the effect can be compared to that ofa known agent.

[0024] The investigation of the role of Component B (CB) in in vivoangiogenesis studied in the rabbit cornea assay was performed by:

[0025] a) testing the ability of the molecule to produce vessel growthwhen placed into the avascular corneal stroma;

[0026] b) testing the ability of the molecule to favour or repressneovascularization elicited by the angiogenesis factor basic fibroblastgrowth factor (bFGF) or vascular endotelial growth factor (VEGF).

[0027] Methods

[0028] Protocol For Slow-release Preparation of Test Compounds

[0029] Growth factors or peptides were prepared as slow-release pellets.

[0030] Slow-release pellets (1×1×0.5 mm) were prepared in sterileconditions incorporating the test substances into a casting solution ofa ethinyl-vinyl copolymer (Elvax40, Dupont, Wilmington, Del.), in 10%methylene chloride (10 μl/droplet) (Langer and Folkman, 1976; Ziche etal. 1982).

[0031] Surgical Procedure

[0032] The angiogenic activity was assayed in vivo using the rabbitcornea assay.

[0033] In the lower half of New Zealand white rabbit eye (Charles River,Calco, Lecco, Italy), anaesthetised by sodium pentothal (30 mg/kg), amicro pocket (1.5×3 mm) was surgically produced using a pliable irisspatula 1.5 mm wide.

[0034] The pellets were implanted in the micropockets located into thetransparent avascular corneal stroma.

[0035] Quantification of Corneal Angiogenesis

[0036] Subsequent daily observations of the implants were made with aslit lamp stereomicroscope without anaesthesia. An angiogenic responsewas scored positive when budding of vessels from the limbal plexusoccurred after 34 days and capillaries progressed to reach the implantedpellet according to the scheme previously reported (Ziche et al. 1989).Angiogenic activity is expressed as the number of implants exhibitingneovascularization over the total implants studied. Potency is scored bythe number of newly formed vessels and by their growth rate. Data areexpressed as angiogenesis score, calculated as vessel density x distancefrom limbus in mm. A density value of 1 corresponded to 0 to 25 vesselsper cornea, 2 from 25 to 50, 3 from 50 to 75, 4 from 75 to 100 and 5 formore than 100 vessels (Ziche et al., 1994).

[0037] Experimental Design

[0038] The effect of Component B was tested following two procedures:

[0039] A) Three different concentrations of the molecule were tested inthe cornea of at least 4 distinct rabbits per each dose, to define thepotential angiogenic activity of the compound. The effect of Component Bwas compared with that elicited by the growth factor bFGF at 50 and 100nag/pellet. In this experimental protocol rabbits were monitored for 3weeks.

[0040] B) To evaluate a potential role of Component B in modulatingangiogenesis the effect of this agent was tested in the presence of adefined angiogenesis factor, i.e. bFGF. To this aim two adjacent pocketswere surgically produced in the same cornea, one bearing the angiogenictrigger and the other Component B. Experiments were also performedtesting both substances incorporated into the same pellet (FIG. 1).

[0041] This last experimental protocol was specifically set up by ourgroup to define:

[0042] 1) the effect of the agent as a “costimulator” of theangiogenesis elicited by bFGF;

[0043] or 2) the ability of the agent to inhibit angiogenesis elicitedby the growth factor (Ziche et al, 1992 and 1994).

[0044] In this experimental protocol the rabbits were monitored for 4-5weeks. The same protocol was used to tests the effect of CB as“costimulator” of the angiogenesis induced by VEGF.

[0045] Histological Analysis

[0046] Rabbit corneas with corneal pockets containing CB and/or bFGFwere sampled at 2, 6, 15 days post surgery, and fixed in formalin afterremoving the pellets. Routine histopathological processing wasperformed; sections 5 μm thick were cut next to where each pellet wasplaced; sections were stained with hematoxylineosin. At least 40sections were examined per each cornea.

[0047] Statistical Analysis

[0048] Results are expressed as means for (n) implants. Angiogenic scoredata contained both positive and negative results. Multiple comparisonswere performed by one-way ANOVA and individual differences were testedby Fisher's test after the demonstration of significant intergroupdifferences by ANOVA. A P value <0.05 was taken as significant (see alsoAppendix for further statistical evaluation).

[0049] Results

[0050] a) Angiogenic Activity of CB

[0051] The angiogenic activity of CB was tested after incorporatingincreasing concentrations of the compound in slow release pellets of thepolymer Elvax-40. Solubilization and incorporation of the compound intothe polymer pellets did not cause any specific problem. The doses testedwere: 0.2, 0.5, 2 and 4 pg/pellet. The effect of CB was compared to thatproduced by basic fibroblast growth factor (bFGF).

[0052] CB elicited a dose-dependent angiogenic effect whose potencyappeared to be weaker than the one elicited by bFGF. In FIG. 2A data arereported on the angiogenic activity of bFGF obtained from previousexperiments and from the experiments run in parallel with CB. In FIG. 2Bdata from daily observation of rabbit corneas implanted withCB-containing pellets are reported as angiogenic score. The highestangiogenic score obtained with CB averaged around 3-3.5 (24 μg/pellet)(P<0.05 vs vehicle pellets alone) vs 7-8 produced by bFGF (0.2μg/pellet)(P<0.05 vs vehicle pellets alone). CB was not angiogenic atthe concentration of 0.2 μg/pellet. As shown in Table 1, 0.5 μg/pelletCB induced a positive angiogenic response in 1 implant out of 5performed. Two and 4 μg/pellet were the most effective doses. Thesedoses induced a similar angiogenic activity and produced 2 positiveimplants out of 5 performed.

[0053] CB was devoid of any macroscopic inflammatory activity asrevealed by the persistence of corneal transparency all through theexperiments at any concentration tested.

[0054] To assess the specificity of CB angiogenic effect, the compoundwas heat-inactivated (h.i.) by boiling it for 20 min. The dose of 2 μgwas then tested. Following heat inactivation CB completely lostangiogenic activity (FIG. 3)(P<0.05 vs CB 2μg).

[0055] b) Effect of CB on the Angiogenesis Induced by bFGF

[0056] To evaluate the potential role of CB in modulating the effect ofa known angiogenic effector, experiments were performed testingsuboptimal concentrations of both substances (500 ng of CB and 100 ng ofbFGF) co-released into the corneal stroma. Experiments were performedtesting both substances incorporated into the same pellet (FIG. 1A).Furthermore the compounds were tested at the same concentration as abovebut released into the stroma separately in 2 independent pellets (FIG.1B).

[0057] The simultaneous presence of CB and bFGF into the corneaincreased the angiogenic response elicited by either individual agent(FIG. 4A and B, Table 2).

[0058] Angiogenesis occurred earlier and progressed more rapidlyproducing a significant increment of the number of newly formed vessels(P<0.05 vs CB and bFGF alone). This effect was apparent in bothexperimental conditions.

[0059] However, when CB and bFGF were released independently by 2separate pellets the effect was higher. Capillaries grew toward bFGFrather than CB suggesting that CB contributed to potentiate bFGFactivity. After 7 days, neovascular growth started to regress.

[0060] Additional experiments were performed with increasingconcentrations of CB (0.2, 0.5 and 2 μg/pellet) on the angiogenesiselicited by a constant concentration of bFGF (100 ng). A synergismbetween the two molecules could be observed (FIG. 5). Interestingly, themost effective condition of synergism between CB and bFGF was observedwith 200 ng CB (P<0.05 vs CB and bFGF alone) tested in two separatepellets.

[0061] c) Effect of CB on the Angiogenesis Induced by VEGF

[0062] In Table 3 the synergistic effect of Component B on VEGF-inducedangiogenesis are reported.

[0063] The synergism between CB and VEGF was evaluated with the factorstested in two separate pellets. The results obtained at day 10 arereported in Table 3. The data are expressed as the number of implantsexhibiting neovascularization with an angiogenesis score equal or overto 6, over the total implants performed.

[0064] Further statistical analysis was performed in order to confirmpossible positive interactions between the test compounds using a moreconservative analysis (see FIG. 11 in Appendix).

[0065] The main factors (“test compounds” and “angiogenic score” overtime) were analysed according to the Multifactor Analysis. The resultsshowed that statistically significant differences (p<0.0001) are presentamong the test compounds over the time.

[0066] As to the interaction among the test compounds the results of theTukey's test allow the following considerations (see FIG. 12 below):

[0067] CB 500 ng+bFGF 100 ng (1 pellet)

[0068] Both compounds individually are not statistically different fromcontrols. The combination of the two compounds gave a response which isstatistically different from either the controls and the single drugs.The response is around the expected additive effect.

[0069] CB 200 ng+bFGF 100 ng (2 pellets)

[0070] Both compounds individually are not statistically different fromcontrols. The combination of the two compounds gave a response which isstatistically different from either the controls and the single drugs.In addition, it should be noted that the response of the combinedtreatments clearly exceeds the expected additive effect. The above seemsto confirm the presence of a synergistic effect between the two drugs.

[0071] CB 500 ng+bFGF 100 ng (2 pellets)

[0072] Both compounds are not statistically different from controls. Thecombination of the two compounds gave a response which is statisticallydifferent either from the controls and the single drugs. The response isaround the expected additive effect. In addition, no differences werefound comparing CB 500 ng+bFGF 100 ng (1 pellet) vs CB 500 ng+bFGF 10 ng(2 pellet).

[0073] CB 2 μg+bFGF 100 ng (2 pellets)

[0074] Both compounds are not statistically different from controls. Thecombination of the two compounds gave a response which is statisticallydifferent from controls but not from the single compounds. The responseis around the expected additive effect.

[0075] Histological Analysis

[0076] The effect of CB was examined at the maximal effectiveconcentration (4 μg) and at a suboptimal concentration (500 ng) in thepresence and in the absence of bFGF (100 ng). No difference in theextent of cellular infiltrate was apparent between CB and bFGF implantsin any combination (FIGS. 7,8,9). Within 2 days from the implants aleukocyte infiltrate surrounded a dense network of newly formedcapillaries in proximity of the limbal region at the epithelial side ofthe cornea (FIG. 10). At day 6 a consistent reduction in the extent ofthe leukocyte infiltrate was apparent while capillary vessels appearedincreased in number and caliber in response to either molecule (FIG.11). At day 15 the extent of the leukocyte infiltrate was negligiblewhile capillaries appeared morphologically unmodified.

[0077] Conclusions

[0078] Component B possesses angiogenic activity which is apparent inthe concentration range of micrograms and which is lost by heatinactivation. Most of the angiogenesis factors are angiogenic atconcentrations 2040 fold lower. Together with the high concentrationrequired to elicit angiogenesis, 2 aspects appear relevant in CB effect:

[0079] 1) the ability to elicit budding of capillaries within the first34 days from the implant, mimicking the secreted angiogenesis factorVEGF rather than the matrix linked angiogenesis factor bFGF;

[0080] 2) the flattening over time of the efficiency of neovasculargrowth, leading to only 30-40% of the implant tested to be fullyvascularized after 10-14 days. Our results indicate that CB inducesangiogenesis in vivo and has the ability to synergize with bFGF inpromoting neovascular growth in the rabbit cornea. These considerationstogether with the characteristics of the potentiation of the angiogenicresponse in the presence of bFGF, suggest that CB requires the presenceof additional growth factors to fully express its angiogenic potentialin vivo.

[0081] Histological examination of corneal sections sampled at varioustime intervals was performed to assess whether the angiogenesis processelicited by CB involved inflammatory cell infiltrate. The effect of CBwas compared to that produced by the corneal implant of bFGF. At routinehistological examination we did not find major differences in the extentand in the type of leukocyte infiltrate in corneas receiving CB, bFGF orthe combination of the two. Thus from our results we can conclude thatthe corneal vascularization induced by CB does not appear to be mediatedby gross inflammatory reaction products since no sign of corneal opacitywas apparent.

[0082] The characteristic of the angiogenic response elicited in theavascular cornea by CB suggests that CB might facilitate some of theearly events required to mobilise endothelial cells from capillaries.Once this process is started and endothelial cells are “loosened” fromthe tight boundary to the extracellular matrix, bFGF expresses itsmitogenic effect with more efficiency. TABLE 1 Effect of Component B onangiogenesis in the rabbit cornea Positive Positive CB implants/totalbFGF implants/total (μg/pellet) performed (ng/pellet) performed 0 0/6 00/6 0.2 0/5 50 1/6 0.5 1/5 100 2/6 2 2/5 200 5/6 4 2/5

[0083] Data are expressed as positive implants exhibitingneovascularization over the total implants performed. The resultsobtained at day 7 are reported.

[0084] An angiogenic response was scored positive when budding ofvessels from the limbal plexus occurred after 3-4 days and capillariesprogressed to reach the pellet containing the angiogenic favors. TABLE 2Synergistic effect of Component B on bFGF-induced angiogenesis PositiveTest compounds implants/total performed CB 500 ng 1/5 bFGF 100 ng 1/5 CB500 ng + bFGF 100 ng (one pellet) 3/6 CB 500 ng + bFGF 100 ng (twopellets) 4/5

[0085] Data are expressed as positive implants exhibitingneovascularization over the total implants performed. The resultsobtained at day 7 are reported.

[0086] An angiogenic response was scored positive when budding ofvessels from the limbal plexus occurred after 3-4 days and capillariesprogressed to reach the pellet containing the angiogenic factors. TABLE3 Synergistic effect of Component B on VEGF-induces angiogenesisPositive implants/total performed CB 200 ng 0/5 CB 500 ng 1/5 VEGF 100ng 1/4 CB 200 ng + VEGF 100 ng 2/4 CB 400 ng + VEGF 100 ng 4/4

1. Use of Component B in combination with a human growth factor for thepreparation of a composition for promoting angiogenesis.
 2. The useaccording to claim 1 in the treatment of wounds, ulcers and othertraumatic lesions to any of the tissues in the body.
 3. Pharmaceuticalcomposition useful as cicatrizants comprising Component B and a humangrowth factor as active principles in combination with apharmaceutically acceptable carrier.
 4. Pharmaceutical compositionaccording to claim 3 wherein the two active principle are both presentin a single administration dose.
 5. Pharmaceutical composition accordingto claim 3 wherein the two active principle are present each in aseparated administration dose.
 6. Pharmaceutical composition accordingto claims 3-5 wherein the human growth factor is bFGF or VEGF.
 7. Methodof treatment of wounds, ulcers and other traumatic lesions to any of thetissues in the body comprising administering in a single administrationdose an effective amount of Component B and an effective amount of ahuman growth factor.
 8. Method according to claim 7 wherein the twoactive principles are administered in separated administration dose. 9.Method according to claims 8 and 9 wherein the human growth factor isbFGF or VEGF.